The Jumonji C domain-containing epigenetic “erasers” are hydroxylases that confer posttranslational modifications on histone tails and can either promote or repress gene transcription. Development of tool molecules that inhibit Jumonji demethylases, facilitate investigations of cancer-associated transcriptional programs. Moreover, the KDM4 enzyme (KDM4A-F) subfamily members include the enzyme KDM4C (also known as JMJD2C), which specifically demethylates di- and trimethylated histone H3 at Lys 9 or Lys 36. Dysregulation of KDM4C has been implicated in prostate, colon, and breast cancer, as this demethylase has been shown to modify the expression levels of various oncogenes. The goal of the present study is to identify a new series of potent and selective small-molecule inhibitors of KDM4C, in potential application as chemical probes. Using high-resolution crystal structures of the KDM4 subfamily members as templates, we have performed a small molecule virtual docking screen. Of the 600,000 fragments docked in silico, 14 fragments were identified for further study. These candidates were assessed in vitro against KDM4C in an enzymatic assay and here we report an overall hit rate of 50%, with 7 compounds exhibiting IC50 values in a range of 18μM to 176μM with ligand efficiency of 0.3 or higher. A subset of molecules containing a 5-aminosalicylate core were selected as candidates for further optimization. This chemotype is of particular interest as these molecules docked to the enzyme in two distinct orientations. These docking poses informed the design of covalently linked fragment compounds, which was followed by several rounds of iterative target-specific compound docking, hybrid molecule design, compound synthesis and further in vitro characterization. Notably, our method demonstrated a substantial increase in potency when we linked the two docked fragments together, through which we have successfully developed a 43nM inhibitor of KDM4C. Seven compounds representing the hybrid inhibitor scaffold have been co-crystallized with KDM4A and superpose well with the docking pose. Finally, several of the hybrid inhibitors are selective for the KDM4 subfamily over the related enzymes including: FIH, KDM2A and KDM6B, while lacking selectivity against the KDM3 and KDM5 subfamily. Currently, we are focusing our efforts on virtual optimizations of inhibitors that show greater selectivity for the KDM4 subfamily.

Virtual fragment screening and linking discovers Jumonji histone demethylase inhibitors

253rd National Meeting (2017)

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